5-Fluorouracil (5-FU) is commonly used in the treatment of cancers, including cancers of the breast, head, neck, and digestive system. The efficacy of 5-FU as a cancer treatment varies significantly among patients. Clinically significant differences in systemic clearance and systemic exposure of 5-FU are often observed. See, Grem, J. L. In Chabner, B. A. and J. M. Collins (eds.), Cancer Chemotherapy: Principles and Practice, pp. 180-224, Philadelphia, Pa., Lippincott, 1990). Furthermore, 5-FU treatment is severely toxic to some patients, and has even caused death. See, Fleming et al. (1993) Eur. J. Cancer 29A: 740-744; Thyss et al. (1986) Cancer Chemother. Pharmacol. 16: 64-66; Santini et al. (1989) Br. J. Cancer 59: 287-290; Goldberg et al. (1988) Br. J. Cancer 57: 186-189; Trumpet al. (1991) J. Clin. Oncol. 9: 2027-2035; and Au et al. (1982) Cancer Res. 42: 2930-2937.
Patients in whom 5-FU is severely toxic typically have low levels of dihydropyrimidine dehydrogenase (DPD) activity. See, Tuchman et al. (1985) N. Engl. J. Med. 313: 245-249; Diasio et al. (1988) J. Clin. Invest. 81: 47-51; Fleming et al. (1991) Proc. Am. Assoc. Cancer Res. 32: 179; Harris et al. (1991) Cancer (Phila.) 68: 499-501; Houyau et al. (1993) J. Nat'l Cancer Inst. 85: 1602-1603; Lyss et al. (1993) Cancer Invest. 11: 239-240. Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the principal enzyme involved in the degradation of 5-FU, which acts by inhibiting thymidylate synthase. See, Heggie et al. (1987) Cancer Res. 47: 2203-2206; Chabner et al. (1989) In DeVita et al. (eds.), Cancer—Principles and Practice of Oncology, pp. 349-395, Philadelphia, Pa., Lippincott; Diasio et al. (1989) Clin. Pharmacokinet. 16: 215-237; Grem et al., supra. The level of DPD activity also affects the efficacy of 5-FU treatments, as 5-FU plasma levels are inversely correlated with the level of DPD activity. See, Iigo et al. (1988) Biochem. Pharm. 37: 1609-1613; Goldberg et al., supra.; Harris et al., supra.; Fleming et al., supra. In turn, the efficacy of 5-FU treatment of cancer is correlated with plasma levels of 5-FU.
In addition to its 5-FU degrading activity, DPD is also the initial and rate limiting enzyme in the three-step pathway of uracil and thymine catabolism, leading to the formation of β-alanine and β-aminobutyric acid, respectively. See, Wasternack et al. (1980) Pharm. Ther. 8: 629-665. DPD deficiency is associated with inherited disorders of pyrimidine metabolism, clinically termed thymine-uraciluria. See, Bakkeren et al. (1984) Clin. Chim. Acta. 140: 247-256. Clinical symptoms of DPD deficiency include a nonspecific cerebral dysfunction, and DPD deficiency is associated with psychomotor retardation, convulsions, and epileptic conditions. See, Berger et al. (1984) Clin. Chim. Acta 141: 227-234; Wadman et al. (1985) Adv. Exp. Med. Biol. 165A: 109-114; Wilcken et al. (1985) J. Inhert. Metab. Dis. 8 (Suppl. 2): 115-116; van Gennip et al. (1989) Adv. Exp. Med. Biol. 253A: 111-118; Brockstedt et al. (1990) J. Inherit. Metab. Dis. 12: 121-124; and Duran et al. (1991) J. Inherit. Metab. Dis. 14: 367-370. Biochemically, patients having DPD deficiency have an almost complete absence of DPD activity in fibroblasts (see, Bakkeren et al., supra) and in lymphocytes (see, Berger et al., supra and Piper et al. (1980) Biochim. Biophys. Acta 633: 400-409. These patients typically have a large accumulation of uracil and thymine in their cerebrospinal fluid (see, Bakkeren et al., supra.) and urine (see, Berger et al., supra.; Bakkeren et al., supra.; Brockstedt et al., supra.; and Fleming et al. (1992) Cancer Res. 52: 2899-2902).
Familial studies suggest that DPD deficiency follows an autosomal recessive pattern of inheritance. See, Diasio et al., (1988) supra. Up to three percent of the general human population are estimated to be putative heterozygotes for DPD deficiency, as determined by enzymatic activity in lymphocytes. See, Milano and Eteinne (1994) Pharmacogenetics. This suggests that the frequency of homozygotes for DPD deficiency may be as high as one person per thousand.
DPD has been purified from liver tissue of rats (see, Shiotani and Weber (1981) J. Biol. Chem. 256: 219-224; Fujimoto et al. (1991); J. Nutr. Sci. Vitaminol. 37: 89-98], pig [Podschun et al. (1989) Eur. J. Biochem. 185: 219-224), cattle (see, Porter et al. (1991) J. Biol. Chem. 266: 19988-19994), and humans (see, Lu et al. (1992) J. Biol. Chem. 267: 1702-1709). The pig enzyme contains flavins and iron-sulfur prosthetic groups and exists as a homodimer with a monomer Mr of about 107,000 (see, Podschun et al., supra.). Since the enzyme exhibits a nonclassical two-site ping-pong mechanism, it appears to have distinct binding sites for NADPH/NADP and uracil/5,6-dihydrouracil (see, Podschun et al. (1990) J. Biol. Chem. 265: 12966-12972). An acid-base catalytic mechanism has been proposed for DPD (see, Podschun et al. (1993) J. Biol. Chem. 268: 3407-3413).
The DPD cDNA is described in copending U.S. application Ser. No. 08/304,309. Recently, DPD mRNA from patients lacking dihydrompyrimidine activity was found to lack an exon which encodes a 165 base pair sequence found in the wild-type DPD cDNA. See, Meinsma et al. (1995) DNA and Cell Biology 14(1): 1-6.
Because an undetected DPD deficiency poses a significant danger to a cancer patient who is being treated with 5-FU, a great need exists for a simple and accurate test for DPD deficiency. Such a test will also facilitate diagnosis of disorders that are associated with DPD deficiency, such as uraciluria. The present invention provides such a test, thus fulfilling these and other needs.